The present invention is directed to a method for treating tissue damage in a mammal caused by ischemia, such as from a stroke or from myocardial ischemia, utilizing a peptidyl diazomethyl ketone.
An ischemia is a deficiency of blood flow to a body part, due to functional constriction or actual obstruction of a blood vessel. Ischemia may occur in various parts of the body of an animal, but it has the most dire consequences when it occurs in the brain, in the heart or in the bowels.
For example, a stroke occurs when there is an interruption of blood flow to an area of the brain. It is usually caused by an embolism or clot that blocks a significant artery in the brain. Blood flow to this area is blocked, causing the affected tissue to become hypoxic. Upon resolution of the cause of the stroke, at least partial blood flow is restored to the affected tissue of the animal. These two processes, lack of blood flow and then restoration of blood flow both produce effects that damage the affected brain tissue.
Among the mechanism of damage initiated by stroke is the activation of calcium sensitive proteases due to the influx of calcium ions into the hypoxic tissue.
At least two classes of proteases, the calpains and the cathepsins, are believed to be activated during cerebral ischemia. Both enzymes are cysteine proteases, the former being located in the cytoplasm, while the latter is located in the lysosome of the cells. Brain tissue contains the cathepsins B, L, S, and H, of which the first two predominate. Moreover, at physiological pH, cathepsin L is less stable than cathepsin B.
Both the calpains and cathepsins play a role in the basal recycling of neural cytoskeletal components. These enzymes are part of a series of enzymes which includes phospholipases, phosphatases, certain kinases and proteases, and which participate in this recycling process.
Cathepsins have been studied under conditions of cardiac ischemia and are believed to play a role in tissue damage by their release from the lysosome into the cytoplasm, where they are able to contribute to cellular injury by the breakdown of cellular structural components. Increased intracellular calcium and acidic pH are believed to be factors leading to lysosomal membrane instability and the activation of these proteases during cardiac ischemia. The same phenomena of calcium influx and acidosis occurs following brain ischemia, and these are believed to cause further cellular damage.
Because tissue perfusion is required for the distribution of pharmaceuticals to target tissues, it is difficult to intervene pharmacologically in the stroke process to prevent the damage caused by hypoxia that occurs during stroke. Thus, the objective of medical research in this area is to reduce the amount of time that the tissue is hypoxic and simultaneously therewith to develop therapies to prevent the activation of the destructive processes that ensues upon restoration of blood flow. In this regard, attention has been focused on the calpains and cathepsins and inhibitors of these two protease classes have been proposed for treating patients afflicted with brain ischemia, myocardial ischemia, and other diseases.
A number of investigators have indeed suggested that calpain inhibition could reduce stroke damage. These cytosolic neutral cysteine proteases are thought to trigger release of the lysosomal proteases. Reduction in tissue damage resulting therefrom has been observed in some experimental systems. However, these results are often complicated by the use of calpain inhibitors that also inhibit a number of other proteases.
Recently, U.S. Pat. No. 4,518,825 to Rasnik, discloses that xcex1-amino fluoroketone peptide derivatives inhibit various proteases, including serine and cysteine proteases, including, but not limited to cathepsins B, H, C, G, R; elastase; trypsin; plasma kallikrein; glandular kallikrein; plasmin; plasminogen activator; and the like. These compounds, however, exhibit non-specific binding to serine proteases, as well as other non-cathepsin cysteine proteases.
Krantz, et al. in U.S. Pat. No. 5,055,451 disclose aryloxy and arylacyloxy methylketones as thiol protease inhibitors, including inhibitors of Cathepsin B. Krantz, et al. recognized that there is a need for potent and specific thiol protease inhibitors and in particular a need for chemically stable inhibitors that minimize the likelihood of non-specific reactions with plasma or cellular constituents. Unfortunately, the aryloxy and arylacyloxy compounds displayed unacceptable toxicity in large animals.
PCT Application WO 96/21655 and U.S. Pat. No. 5,691,368 to Peet disclose oxazolidine inhibitors of calpain and/or cathepsin B; it is alleged that the compounds are useful in the treatment of patients afflicted with acute or chronic neurodegenerative disorders.
EPO Application 525,420 to Ando, et al. disclose peptidyl aminomethylketones that exhibit reversible inhibition of calpains and cathepsins. They allege that these compounds are clinically useful in the treatment of various diseases, including stroke.
It is to be noted that in each of these references, the compounds disclosed therein inhibit a number of enzymes, including but not limited to both cathepsins and calpains. None of the references disclose inhibitors that are specific for only one type of cysteine protease. However, as recognized by both Rasnik and Krantz, et al. described hereinabove, for a drug in this field to be effective, it must not only interact with one type of cysteine protease, but, in addition, there must also be a lack of interaction with other cysteine proteases to prevent unwanted side effects.
The present, inventors, however have found such a system. In particular, they have found that peptidyl diazomethyl ketones, and in particular, N-terminus amino protected diazomethyl ketones such as benzyloxy-carbonyl peptidyl diazomethyl ketones, are capable of specifically inhibiting cathepsins significantly better than calpains.
Benzyloxycarbonyl peptidyl diazomethyl ketones, however, are not new compounds, but have been described in earlier publications by Shaw, et al. For example, Shaw, et al. in Arch. Biochem. Biophys, 1983, 222, 424-429 reveal a number of benzyloxycarbonyl-Phe-X-CHN2 derivatives, wherein X is one of several amino acids or derivatized amino acids and their effect on the in vitro activity of bovine spleen cathepsins. However, these compounds were not considered viable clinical candidates for treating damaged tissues because, as indicated by Powers in U.S. Pat. No. 5,610,297, these compounds are thought, inter alia, to be poorly membrane permeant and to have low specificity.
But, the present inventors have surprisingly found that these compounds are not poorly membrane permeants and do not have low specificity.
Accordingly, the present invention is directed to treating tissue damage in a patient caused by ischemia comprising administering to said patient a therapeutically effective amount of a compound which is an inhibitor of at least one of cathepsin B or cathepsin L, wherein the inhibition of cathepsin B or L is significantly greater than that of calpain, said compound being a peptidyl diazomethyl ketone and more preferably a N-terminus amino protected peptidyl diazomethyl ketone.
The present invention is directed to a method for treating tissue damage in a mammal resulting from ischemia. The compounds useful for this purpose are small lipophilic molecules which are specific inhibitors of cathepsin B or cathepsin L or both. They may inhibit cathepsin B, but not cathepsin L or vice versa or they may inhibit both enzymes. However, it is a significantly poorer inhibitor of calpain relative to either cathepsin B or cathepsin L.
Another characteristic of the present invention is that the compounds utilized are peptidyl diazomethyl ketones. These compounds are preferably di or tri-peptides. In addition, they have a molecular weight ranging from about 450 to about 1000 daltons.
A preferred embodiment of the present invention has the formula:
Zxe2x80x94AA1xe2x80x94AA2xe2x80x94(AA3)nCHN2xe2x80x83xe2x80x83I
wherein
Z is H or an amino protecting group at the N-terminus;
n is 0 or 1;
AA1 is a hydrophobic amino acid residue;
AA3 is a hydrophobic amino acid residue, a hydroxy or thiol containing amino acid or a ether or thioether containing amino acid; and
AA2 is xcex1-amino isobutyric acid or a hydrophobic amino acid residue when n is 1 but when n is 0, AA2 is a hydrophobic amino acid residue, a hydroxy or thiol containing amino acid or an ether or thioether containing amino acid.
A more preferred embodiment of the present invention has Formula I, wherein
Z, n and AA1 are as defined hereinabove;
AA3 is a hydrophobic amino acid residue, AA5 (OR12) or AA5 (SR12) ; and
AA2 is xcex1-amino butyric acid or a hydrophobic amino acid residue when n is 1; but when n is 0, AA2 is a hydrophobic amino acid residue or AA4(OR11) or AA4(SR11) or Met, wherein
AA5 and AA4 are the same or different amino acids which have the OH group or thiol group or thiomethyl group on the side chain is removed;
R11 and R12 are independently hydrogen, lower alkyl, aryl, aryl lower alkyl, heterocyclic or heterocyclic lower alkyl.
An even more preferred embodiment has Formula I
Zxe2x80x94AA1xe2x80x94AA2xe2x80x94(AA3)nCHN2xe2x80x83xe2x80x83I
wherein
Z is H or an amino protecting group at the N-terminus;
n is 0 or 1;
AA1, AA2 and AA3 are amino acid residues;
AA1 is a hydrophobic amino acid residue;
AA3 is a naturally occurring hydroxy or thiol containing amino acid residue or hydrophobic acid;
AA2 is xcex1-amino isobutyric acid (Aib) or a hydrophobic amino acid residue when n is 1; but when n is 0, AA2 is Met, Cit, Aib, Ala, Trp, Ser(OR4), Thr(OR5), Tyr, hTry, Tyr(R9) (OR6), hTyr(R9) (OR6), Cys(SR7), hCys(SR7), hPhe(R8), hSer(OR4) or Phe(R8);
hCys is homocysteine;
hPhe is homophenylalanine;
hSer is homoserine;
hTyr is homotyrosine;
R4, R5, R6 and R7 are independently hydrogen, aryl, aryl lower alkyl, bulky alkyl group or lower alkyl,
R8 is hydrogen or an electron withdrawing group; and
R9 is a substituent on the phenyl ring of the tyrosine and is hydrogen or an electron withdrawing group.
As used herein, the term xe2x80x9camino acid residuexe2x80x9d refers to an amino acid less the hydrogen atom on the N-terminus and less the hydroxy group on the C-terminus end thereof. Various designations, such as the 3 letter or 1 letter abbreviations, have been utilized to represent the amino acids. When these abbreviations are used, it is to be understood that these are abbreviations of amino acid residues, as defined herein. Thus, for example, Ala is 
It is preferred that the amino acid is a natural amino acid. It is also preferred that the amino acids are xcex1-amino acids.
The amino acids contemplated to be used therein include the natural amino acids and derivatives thereof. More specifically, the amino acids utilized herein may be analogs or homologs of the natural amino acids. For example, the side chain may contain alkylene groups bridging the alpha carbon atom on the amino acid and the side chain. The bridging alkylene groups may be lower alkylene containing 1-6 carbon atoms. Preferably, these bridging alkylene groups contain 1-3 carbon atoms and most preferably contain 1 carbon atom. For example, the naturally occurring side chain of phenylalanine is benzyl. A homolog contemplated and to be within the scope of the amino acid utilized in the present invention is a bridging alkylene group containing 1-3 carbon atoms, linking the xcex1-carbon of the amino acid with the benzyl group. If the bridging alkylene group is a methylene A group, it is a homoamino acid. Examples of homologs of amino acids contemplated by the present invention include homoserine, homotyrosine, homocysteine, homophenylalanine, and the like. If the amino acid contemplated is a homoamino acid, it is designated as xe2x80x9chAA.xe2x80x9d
The amino acid residues, especially those containing aromatic as well as heterocyclic groups may be unsubstituted or be substituted with lower alkyl, lower arylalkyl, aryl, lower alkoxy, or hydroxy or electron withdrawing groups, as defined herein.
In the abbreviations described herein, the notation AA(YR) or its equivalent where Y is 0 or S, refers to an amino acid having an OR or SR group on the side chain, which side chain, as defined by the present invention, may be a hydroxy group, ether, thiol, or thioether. In these notations, the AA residue of AA(YR) refers to the amino acid where its side chain is less the hydroxy group, or thiol group, the thioether or ether group. Thus, AAYR refers to the amino acid wherein the YR group replaced the hydroxy, thio, thioether or ether group originally present. For example, in Ser(OBz), Ser refers to a serine moiety, less the hydroxy group on the side chain of the natural serine moieties and wherein the hydroxy group is replaced by benzyloxy. It is preferred each R is independently H, loweralkyl, aryl, lowerarylalkyl, heterocyclic or lowerheterocyclic alkyl.
The group Ser(OR) or Tyr(OR) or its request refers to a serine or tyrosine group in which the side chain contains a hydroxy or ether group. The group Cys(SR)or its equivalent refers to cysteine in which the side chain contains a thiol or a thioether. In addition, the group Phe(R8) or Tyr(R9) refers to a R8 group or R9 group, respectively, as defined herein, substituted on the phenyl ring of phenylalanine or tyrosine, respectively.
As defined herein, xe2x80x9ca hydrophobic amino acidxe2x80x9d is an amino acid containing a lower alkyl or aryl consisting of carbon and hydrogen atoms or aryl lower alkyl as the side chain. It is preferably a naturally occurring amino acid or homologs thereon, especially those homologs having 1-3 additional carbon atoms in the main side chain. The naturally occurring hydrophobic amino acids include such amino acids as Valine, Leucine, Isoleucine, Alanine, Tryptophan and Phenylalanine. Examples of hydrophobic homologs of amino acids include homophenylalanine and the like.
The term xe2x80x9chydroxy containing amino acidsxe2x80x9d or xe2x80x9cthiol containing amino acidxe2x80x9d refers to those naturally occurring amino acids having a hydroxy group or thiol group, respectively, on the side chain. The hydroxy group is connected to the xcex1-carbon on the main chain of the amino acid by a bridging group which is preferably lower alkylene and lower arylalkylene, such as, for example, benzyl. Examples include serine, threonine, cysteine, tyrosine, homoserine, homocysteine, homotyrosine and the like.
The term xe2x80x9caromatic containing amino acidxe2x80x9d refers to the naturally occurring amino acids having an aromatic group on the side chain. By aromatic, it is meant an aryl moiety containing only carbon ring atoms. It is preferred that the aromatic group contains 6-10 ring carbon atoms. It excludes heteroaromatics. The aromatic groups may be unsubstituted or substituted with electron withdrawing groups, as defined hereinbelow, or such electron donating groups as hydroxy, lower alkoxy, aryloxy, lowerarylalkoxy, or alkyl, aryl, or aryl lower alkyl. Preferably, the aromatic ring may be unsubstituted or substituted with various groups, such as alkyl, aryl, aryl lower alkyl, halo, hydroxy, lower alkoxy, aryloxy, lower aryl alkoxy, and the like. The alkyl groups, when used singly or in combination with respect to the substituents on the aromatic ring are preferably lower alkyl groups, as defined hereinbelow. Moreover, the term xe2x80x9carylxe2x80x9d, as used herein is as defined hereinbelow. Examples of aromatic containing amino acids include Tyrosine, Tryptophan, and Phenylalanine.
As defined herein, when the amino acid residue has a hydroxy containing or thiol containing side group, the thiol or hydroxy group may be maintained or the hydrogen atom of the hydroxy or thio group is replaced with an aryl group, aryl lower alkyl group, an alkyl group, as defined hereinabove or a bulky alkyl group, thereby forming an ether or thioether, respectively.
As defined herein, the term xe2x80x9clower alkyl groupxe2x80x9d when used singly or in combination, refers to an alkyl group containing 1-6 carbon atoms. Examples include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, t-butyl, isopropyl, pentyl, neopentyl, hexyl, and the like.
The term xe2x80x9carylxe2x80x9d refers to an aromatic moiety containing 6-14 ring carbon atoms and up to a total of 20 carbon atoms. Examples include, phenyl, tolyl, xylyl, naphthyl, and the like.
xe2x80x9cAryl lower alkylxe2x80x9d signifies an alkyl group which is bridged to the main chain by a lower alkylene group as defined herein. Examples include benzyl, phenethyl and the like.
As used herein, the term xe2x80x9cbulky alkyl groupxe2x80x9d refers to a tertiary alkyl group. Preferably it contains 4-10 carbon atoms, and more preferably 4-6 carbon atoms. Examples include tertiary butyl, neopentyl, 2,2-dimethylbutyl, and the like.
The term xe2x80x9celectron withdrawing groupxe2x80x9d refers to a group which is more electronegative than hydrogen. Examples include nitro, cyano, halo, amido, carboxy, carboloweralkoxy, sulfonyl, sulfinyl, and the like, with the most preferred being a nitro or halo group.
The term heterocyclic, when used singly or in combination refers to a cyclic ring which may be saturated partly unsaturated or an heteroaryl and contain one, two or three hetero ring atoms. The heterocyclic rings include the benzoheterocyclics. The heterocyclic ring contain from 5-14 ring atoms. If it is preferred that the heterocyclic group contains 1, 2 or 3 ring heteroatoms selected from N, S or 0 and contain at least 2 carbon ring atoms and up to a total of 13 ring carbon atoms and up to a total of 18 carbon atoms. It is preferred that the hetercyclic ring is monocyclic or bicyclic and contains 5-10 ring atoms. Typical examples include thienyl, furyl, tetrahydrofuryl, oxazolyl, benzoxazolyl, pyrrolyl, pyridyl, imidazoyl, benzothienyl, pyranyl, pyrazolyl, pyrazinyl, indolyl, pyrimidinyl, isoquinolyl, quinolyl, piperidyl, morpholinyl, indolinyl, and the like.
As used herein, Z is an amino protecting group on the N-terminus end. Although it is not necessary that a protecting group on the N-terminus end be present (i.e., Z may be H), it is preferred that Z is a protecting group. These protecting groups block the amino group on the N terminal end of the amino acid or peptide.
A number of blocking reagents for amino groups are known in the art. Examples of amino protecting groups are described in an article entitled xe2x80x9cSolid Phase Peptide Synthesisxe2x80x9d, by G. Barany and R. B. Merrifield in THE PEPTIDES, Vol. 2, edited by E. Gross and J. Meienhoffer, Academic Press, N.Y., N.Y. 100-118 (1980) and in the book entitled xe2x80x9cPROTECTING GROUPS IN ORGANIC SYNTHESISxe2x80x9d by T. W. Green, John Wiley and Sons, New York, the contents of both of which are being incorporated by reference.
Examples of blocking groups include 9-lower alkyl-9-fluorenyloxycarbonyl, 2-chloro-1-indanylmethoxy-carbonyl (CLIMOC), benz[f]indene-3-methyloxycarbonyl (BIMOC), 2-(t-butyl sulfonyl)-2-propenyloxycarbonyl (Bspoc), benzothiophene sulfone-2-methoxycarbonyl (Bsmoc), t-butyloxycarbonyl (BOC), t-amyloxycarbonyl (Aoc), xcex2-trimethylsilylethyloxycarbonyl (TEOC), adamantyl-oxycarbonyl (Adoc), 1-methylcyclobutyloxycarbonyl (Mcb), 2-(p-biphenylyl)propyl-2-oxycarbonyl (Bpoc), 2-(p-phenylazophenyl)propyl-2-oxycarbonyl (Azoc), 2,2-dimethyl-3,5-dimethyloxybenzyloxycarbonyl (Ddz), 2-phenylpropyl-2-oxycarbonyl (Poc), benzyloxycarbonyl (Cbz), Phthaloyl, piperidine-oxycarbonyl, formyl, acetyl, nicotinyl, i.e., 
It is preferred that a lipophilic amino protecting group is used, i.e., the protecting group contains a lipophilic moiety. For example, it is preferable that a moiety of the formula 
wherein R10 is a lipophilic group, i.e., it is substantially non-polar. For example, R10 is hydrocarbyl (containing only C and H atoms), silyl hydrocarbyl, (containing Si, C and H), or heterocyclic (containing C and H and ring N, O, or S atoms, containing up to 14 ring atoms, and 1, 2 or 3 ring heteroatoms, and 3-13 ring carbon atoms), including heteroaromatic. The blocking group, if present, is preferably sufficiently lipophilic to permit the compounds utilized in the present invention to diffuse or penetrate the cell membrane. It is especially preferred that Z is Cbz.
The inhibitors used in the present invention are preferably compounds of Formula I described hereinabove. The compounds of Formula I can also be depicted as below in Formula Ia:

wherein Z and n are as defined hereinabove and
R1 R2 and R3 are independently side groups of amino acids, wherein R1 refers to the side group of the amino acid of AA1 as defined hereinabove, R2 is the side group of the amino acid AA2 as defined hereinabove and R3 is the side group of the amino acid AA3 as defined hereinabove.
It is preferred that AA1 is a naturally occurring amino acid residue. Preferred values of AA1 are Phe, hPhe, Val or Leu and most preferably Phe. When present, the preferred AA3 is Serine, Threonine, Cysteine, homocysteine or homoserine, homotyrosine and especially tyrosine.
The preferred AA2 is a hydrophobic naturally occurring amino acid. When n is 1, the preferred AA2 is Leu; but when n is 0, it is preferred that AA2 is Cit, Ala, Phe, hPhe, Thr (OR5), Phe(R8) hPhe(R8), Aib, Met, Trp, Tyr(OR6), Ser(OR4), hSer(OR6) or Val, wherein R5 and R6 are independently H, aryl (e.g. phenyl), lower aryl alkyl (e.g., benzyl) and especially bulkyl alkyl (e.g., t-butyl), R4 is H, aryl (e.g. phenyl) and especially aryl alkyl (e.g., benzyl), and R8 is H or an electron withdrawing substituent listed hereinabove, especially nitro or halo (e.g., I). With respect to R8, it is preferred that the electron withdrawing group be in the ortho (2-position), and especially para (4-position) positions of the phenyl ring of the phenylalanine. It is more preferred that AA2 is solely a hydroxy containing or thiol containing amino acid, and most preferably a hydroxy containing amino acid, especially tyrosine and serine which is unsubstituted or substituted as defined hereinabove.
In a preferred embodiment, n is 0.
It is more preferred that the compounds utilized in the invention have the formula:
Zxe2x80x94Phexe2x80x94AA2xe2x80x94CHN2
wherein AA2 is as defined hereinabove and more preferably is Ser(OR4), Thr (OR5), or Tyr(OR6), hSer(OR4) or hTyr(OR6),
wherein R4, R5 and R6 are independently hydrogen, aryl, lower aryl alkyl or bulky alkyl and Z is a lipophilic amino protecting group.
The preferred R4 value is aryl or lower aryl alkyl, especially benzyl.
It is preferred that R5 and R6 are bulky alkyl, e.g., neopentyl and especially t-butyl.
Preferred embodiments of the present invention include benzyloxycarbonyl-Phe-Ser(OBz)-CHN2 (also known as CP-1), benzyloxycarbonyl-Phe-Tyr(OtBu)-CHN2 (also known as CP-2), benzyloxycarbonyl-Phe-Cys-(SBz)-CHN2, benzyloxycarbonyl-Phe-Cit-CHN2, benzyloxycarbonyl-Phe-Ala-CHN2, benzyloxycarbonyl-Phe (I) -Ala-CHN2, benzyloxycarbonyl-Phe-Phe-CHN2, benzyloxycarbonyl-Phe-Thr(O-t-Bu)-CHN2, benzyloxycarbonyl-Phe-Phe (p-N02) -CHN2, benzyloxycarbonyl-Phe-Aib-CHN2, benzyloxycarbonyl-Leu-Met-CHN2. benzyloxycarbonyl-Leu-Trp-CHN2, benzyloxycarbonyl-Leu-Tyr-CHN2, benzyloxycarbonyl -Leu-Leu-Tyr-CHN2, and benzyloxycarbonyl-Val-Val-CHN2, wherein Bz is benzyl, Aib is amino-isobutyric acid and Cit is citruilline.
The most preferred embodiments are CP-1 and CP-2.
Since the compounds utilized in the present invention are comprised of amino acids, and since each of the amino acids contain at least one chiral carbon atom, each amino acid moiety can exist in the D or L form. Thus, the compounds utilized in the present invention can exist in various stereoisomeric forms, including enantiomers, diastereoisomers, a combination thereof, as well as racemic mixtures. The use of all of these stereoisomers is contemplated to be within the scope of the present invention.
The compounds used in the present invention are either commercially available or are prepared by art recognized methods. An exemplary procedure is as follows:
For example, the compounds of the present invention are prepared by reacting the mixed anhydride of the corresponding peptide with diazomethane in an inert solvent, under conditions sufficient to form the diazomethyl ketone. The solvent utilized is inert to the peptide, the mixed anhydride thereof, reagents utilized to prepare the mixed anhydride and the product. Examples of the solvent that can be utilized include ether, especially lower alkyl ethers, such as diethyl ether, tetrahydrofuran, and the like. In an embodiment, the diazomethane in the inert solvent is added to the mixed anhydride. Inasmuch as diazomethane is quite reactive, the reaction is conducted at low temperatures, however, the temperature is sufficiently high to effect formation of the product but at a temperature lower than about 10xc2x0 C. It is preferred that the reaction be conducted at a temperature at or below 50C.; and more preferably at a temperature at or above about xe2x88x92500xc2x0 C. and at or below 5xc2x0 C. It is more preferred that the reaction be initially conducted at about xe2x88x9225xc2x0 C. up to a temperature of about 0C. In addition, it is preferred that the diazomethane be added dropwise to the mixed anhydride.
The mixed anhydride is prepared by reacting the peptide with an anhydride forming reagent, such as an acid halide, (e.g., an acid chloride) of a carboxylic acid containing 1-6 carbon acids, or lower alkyl ester thereof. An example of an anhydride forming reagent is isobutyl chloroformate.
The peptide is prepared by methodology-known in the art such as by, for example, reacting under amide forming conditions a first amino acid having a protecting group on the C-terminus and a second amino acid or acid halide thereof or ester thereof e.g., N-hydroxy succinimide ester, having a protecting group thereon, such as a benzyloxycarbonyl protecting group (Z-amino acid). A dehydrating agent, such as dicyclohexyl carbodiimide, may also be present. The protecting group on the C-terminus is then removed by methods known in the art. Examples of C-terminus protecting groups are known in the art and is described in the text, Protective Groups in Organic Synthesis, by Theodora W. Greene, John Wiley and Sons, 1981 (xe2x80x9cProtective Groupsxe2x80x9d) the contents of which are incorporated by reference. If a peptide larger than a dipeptide is desired, then the dipeptide described hereinabove is reacted under amide forming conditions with a third amino acid or acid derivative thereof, (e.g., halide, ester, and the like) in which the C-terminus is either unprotected or protected with a blocking group known in the art, e.g., a blocking group described in xe2x80x9cProtective Groupsxe2x80x9d. The process is repeated until the desired peptide is obtained. Alternatively in compounds comprised of tripeptides or higher, the smaller peptide units are prepared in a manner similar to that described hereinabove, and then the smaller peptide unit is reacted with an amino acid or acid derivative or another peptide or acid derivative under amide forming conditions, in accordance with the procedure described hereinabove, to form the desired higher peptide. Alternatively, the peptide is prepared by a combination of the procedures described herein.
As an illustration of the procedure, Z-AA1xe2x80x94Osu, wherein Osu is the N-hydroxysuccinimide ester and Z is an amino protecting group, such as benzyloxycarbonyl, is reacted with H2Nxe2x80x94AA2xe2x80x94OH in an equimolar ratio in DMF, triethylamine and dimethoxyethane under amide forming conditions. The product thereof is converted to the mixed anhydride and reacted with etheral diazomethane and after work-up, the corresponding benzyloxycarbonyl peptidyl diazomethyl ketone compound is isolated.
The compounds described herein are administered to a patient in need thereof in therapeutically effective amounts.
As used herein, the term xe2x80x9cpatientxe2x80x9d refers to a warm blooded animal such as a mammal which has tissue damage caused by ischemia. It is understood that guinea pigs, dogs, cats, rats, mice, horses, cattle, sheep, and humans are examples of animals within the scope of the meaning of the terms xe2x80x9cmammalxe2x80x9d and xe2x80x9cpatientsxe2x80x9d; it is preferred that the patient is human.
The term xe2x80x9ctherapeutically effective amountxe2x80x9d refers to an amount which is effective, upon continuous infusion or upon single or multiple dose administration to the patient, in providing a reduction in the extent of damage resulting from ischemia, leading to an improved outcome and/or a delay or prevention of disease progression as compared to outcomes expected or obtained in the absence of treatment. The term xe2x80x9ctherapeutically effective amountxe2x80x9d does not necessarily indicate a total elimination or cure of the tissue damage. In determining the therapeutically effective amount or dose, a number of factors are considered by the attending diagnostician, including, but not limited to: the species of mammal; its size, age, and general health; the specific disease involved; the degree of or involvement or the severity of the disease; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regiment selected; the use of concomitant medication; and other relevant circumstances. Based upon these factors, it is within the purview of the ordinary skilled artisan to determine the therapeutically effective amount to be administered to the patient.
A therapeutically effective amount of the compound described herein preferably ranges from about 0.01 milligram per kilogram of body weight per day (mg/kg/day) to about 100 mg/kg/day. Preferred amounts are expected to vary from about 0.5 to about 30 mg/kg/day.
In effecting treatment of a patient afflicted with a disease state described above, a compound utilized in accordance with the present invention can be administered in any form or mode which makes the compound bioavailable in effective amounts, including oral and parenteral routes. For example, it can be administered orally, subcutaneously, intramuscularly, intravenously, transdermally, intranasally, rectally, topically, and the like. Oral or intravenous administration is generally preferred. One skilled in the art of preparing formulations can readily select the proper form and mode of administration depending upon the particular characteristics of the compound selected for the disease state to be treated, the stage of the disease, and other relevant circumstances. The compounds can be administered alone or in the form of a pharmaceutical composition in combination with pharmaceutically acceptable carriers or excipients, the proportion and nature of which are determined by the solubility and chemical properties of the compound selected, the chosen route of administration, and standard pharmaceutical practice. The compounds described herein, while effective themselves, may be formulated and administered in the form of their pharmaceutically acceptable salts, such as for example, acid addition salts, for purposes of stability, convenience of crystallization, increased solubility and the like.
The pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art. The carrier or excipient may be a solid, semi-solid, or liquid material which can serve as a vehicle or medium for the active ingredient. Suitable carriers or excipients are well known in the art. The pharmaceutical composition may be adapted for oral, parenteral, or topical use and may be administered to the patient in the form of tablets, capsules, suppositories, solution, suspensions, or the like.
The compounds described herein may be administered orally, for example, with an inert diluent or with an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like. These preparations should contain at least 4% of the compound, i.e., the active ingredient, but this amount may be varied, depending upon the particular form and may conveniently be between about 4% to about 70% of the weight of the unit. The amount of the compound present in the pharmaceutical compositions is such that a suitable dosage will be obtained. It is preferred that an oral dosage unit form contains between about 5.0 and about 300 milligrams of the peptidyl diazomethyl ketones described herein.
The tablets, pills, capsules, troches and the like may also contain adjuvants typically utilized in the preparation of pharmaceuticals. They may include one or more of the following adjuvants: binders such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose, disintegrating agents such as alginic acid, corn starch and the like; lubricants such as magnesium stearate or zinc stearate; glidants such as colloidal silicon dioxide; and sweetening agents such as sucrose or saccharin may be added or a flavoring agent such as peppermint, methyl salicylate or orange flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or a fatty oil.
Other dosage unit forms may contain other materials which modify the physical form of the dosage unit, for example, as coatings. Thus, tablets or pills may be coated with sugar, shellac, or other enteric coating agents. A syrup may contain, in addition to the peptidyl diazomethyl ketone compounds described herein, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.
For the purpose of parenteral therapeutic administration, the compounds may be incorporated into a solution or suspension. These preparations should contain at least 0.1% of the peptidyl diazomethyl ketone compound described herein, but the amount may be varied to be between 0.1 and about 50% of the weight thereof. The amount of the compound present in such compositions is such that a suitable dosage will be obtained. Preferred compositions and preparations are prepared so that a parenteral dosage unit contains between about 5.0 to about 100 milligrams of the active compound.
The compounds may also be administered topically, and when done so the carrier may suitably comprise a solution, ointment or gel base. Again, they are comprised of components normally utilized in preparing these types of pharmaceuticals. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bees wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Topical formulations may contain a concentration of the compound or its pharmaceutical salts from about 0.1 to about 10% w/v (weight per unit volume).
The solutions or suspensions comprise ingredients normally utilized in these types of pharmaceuticals. For example, they may also include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylene diaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.
As used herein, the singular shall include the plural and vice versa.
The preferred xe2x80x9chydrocarbylxe2x80x9d groups contain 1-20 carbon atoms.
Unless indicated to the contrary, the term xe2x80x9cpeptidyl diazomethyl ketonexe2x80x9d refers to the free as well as the N-amino protected peptidyl diazomethyl ketone.
Unless indicated otherwise, percentages are by weight.